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. 2007 Jun;27(12):4589-600.
doi: 10.1128/MCB.02027-06. Epub 2007 Apr 2.

The ancient mariner sails again: transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends

Csaba Miskey  1 , Balázs Papp, Lajos Mátés, Ludivine Sinzelle, Heiko Keller, Zsuzsanna Izsvák, Zoltán Ivics
Affiliations

The ancient mariner sails again: transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends

Csaba Miskey et al. Mol Cell Biol. 2007 Jun.

Abstract

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage approximately 50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution.

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Figures

FIG. 1.
FIG. 1.
Structure of Hsmar1 transposons in the human genome and transpositional activities of consensus and reconstructed ancestral transposases. (A) Summary of the structures and copy numbers of Hsmar1-derived sequences. The arrows represent IRs. SETMAR is a chimeric protein made up of a histone methyltransferase (SET) domain and a particularly conserved transposase (Tpase) domain. (B) The inset summarizes the principles of a transposition assay in tissue culture. The transposon containing a selectable antibiotic resistance gene (neo) is transfected either with or without a transposase- expressing helper plasmid. Transfected cells are placed under antibiotic selection. The dramatic increase in the number of resistant cell colonies in the presence of transposase is the result of transposition of the element from the plasmid vector into chromosomes. The diagram shows the transpositional activity of Hsmar1-Ra in human HeLa cells. The pHsmar1-neo donor plasmid was cotransfected with pCMV-Hsmar1 consensus, pCMV-SB (expressing the Sleeping Beauty transposase), and pCMV-Hsmar1-Ra. The efficiency of transgene integration was determined by counting G418-resistant colonies. The numbers on the y axis represent mean values obtained from at least three independent experiments. (C) Alignment of mariner transposase amino acid sequences of the cecropia subfamily in the region of the four predicted amino acid substitutions (asterisks). The alignment revealed that the changes predicted in silico correspond to the majority-rule consensus amino acid sequence at these positions. (D) In vitro DNA-binding activity of the Hsmar1-Ra transposase. A radioactively labeled DNA fragment of 83 bp containing the 5′ IR of the Hsmar1 transposon (depicted below) was incubated with the purified MBP-Hsmar1-Ra protein, and the DNA-protein complexes were visualized by EMSA autoradiography. Compared to free, unbound probe (lane 1), increasing concentrations of MBP-Hsmar1-Ra (lanes 2 to 4) produced DNA-protein complexes. Complex assembly was challenged with cold (unlabeled) probe as a specific competitor (lanes 5 to 7), or increasing amounts of pBluescript were used as nonspecific competitor DNA (lanes 8 to 10).
FIG. 2.
FIG. 2.
Excision and integration of Hsmar1. (A) Transposon excision assay in cultured cells. The two functional components of the transposon system (a transposon and a transposase source) are introduced into cells. The transposase catalyzes transposon excision; Tc1/mariner elements leave behind a 2- to 4-nucleotide-long 3′ overhang at the DSBs. DNA repair rejoins the ends of the broken DNA, and the resulting sequences can be recovered by PCR using primers flanking the excision site. (B) Agarose gels showing PCR products of excision assays obtained from HeLa cells and zebra fish embryos. The appearance of the 260-bp bands in the presence of pCMV-Hsmar1-Ra (lane 1) or Hsmar1-Ra mRNA (lane 3) indicates transposon excision from the pHsmar1-neo donor plasmids, followed by excision site repair. Transfection of pCMV-SB (lane 2) and injection of SB mRNA (lane 4) served as controls. M, 250-bp size marker. (C) Hsmar1 transposon footprints identified from cloned excision PCR products from HeLa cells and zebra fish embryos. The sequence of the donor plasmid is schematically shown above. Flanking TA dinucleotides are in boldface, and footprints are in uppercase letters. (D) Three human chromosomal sequences that served as target sites for Hsmar1 transposons. Duplicated TA target dinucleotides are in boldface. (E) Distribution of endogenous genomic Hsmar1 copies (gray columns) and 47 de novo integrants (black columns) in human chromosomes. Mapping of transposon insertions was done using BLAST on the human genome sequence database at NCBI. The graph shows the genomic distribution of transposon insertions with respect to human genes, nongene regions, and repetitive sequences.
FIG. 3.
FIG. 3.
Hsmar1 can function as an autonomous transposon. A schematic representation of the luciferase reporter construct in p5′ UTR/Luc compared to the structure of an Hsmar1 transposon is depicted. (A) Reporter gene expression in HeLa cells from the plasmids indicated below. The error bars indicate standard deviations. (B) Transposon excision assay as shown in Fig. 2. HeLa cells were transfected with the pHsmar1-neo donor plasmid, together with the plasmids indicated. The presence of the characteristic PCR band indicates that the complete Hsmar1 element (Hsmar1-Full) can autonomously catalyze its own excision from the donor plasmid. M, 250-bp size marker.
FIG. 4.
FIG. 4.
Excision of Hsmar1 and MiHsmar1 transposons. (A) Outline of the excision assay. A transposon (Tpson) insertion disrupts and inactivates the Amp resistance gene (Amp). Transposon excision, followed by DNA repair, can restore Amp resistance, which can be selected for in E. coli. (B) Coding triplets of the original, the modified, and the repaired excision sites are listed. Transposon footprints and amino acid sequences are uppercase. Various transposon donor plasmids (indicated on the x axis) were transfected into HeLa cells either with pCMV-Hsmar1-Ra (experiments 1 to 3, numbered below the graph), alone (experiments 4 and 5), or with pCMV-SB (experiment 6) as a control. Indicator plasmids contain the following transposons in the Amp resistance gene: pAmpMITE, the consensus MiHsmar1 sequence; pAmpneo, the neo-tagged Hsmar1 element; pAmpFull, the autonomous Hsmar1 transposon; pAmpneoL, the neo-tagged Hsmar1 element with IR sequences that match those of MiHsmar1; and pAmpFullL, the autonomous Hsmar1 transposon with IR sequences that match those of MiHsmar1. The Ampr/Zeor colonies represent excision events followed by canonical footprint formation at the excision site. The normalized numbers of the double-resistant colonies (shown above the columns) were obtained by dividing the numbers of Ampr/Zeor colonies with the corresponding Zeor colony numbers.
FIG. 5.
FIG. 5.
In vitro DNA binding activity of the SETMAR protein. A radioactively labeled DNA fragment of 83 bp containing the 5′ IR of Hsmar1 (depicted below) was incubated with purified MBP-SETMAR protein, and the DNA-protein complexes were visualized by EMSA. Compared to free, unbound probe (lane 1), increasing concentrations of MBP-SETMAR (lanes 2 to 4) produced DNA-protein complexes. Substantial amounts of the SETMAR protein formed aggregates at all concentrations used and were unable to enter the gel during electrophoresis. Complex assembly was challenged with cold (unlabeled) probe as a specific competitor (lanes 5 to 7), or increasing amounts of pBluescript were used as nonspecific competitor DNA (lanes 8 to 10).
FIG. 6.
FIG. 6.
In vitro cleavage site analysis with ligation-mediated PCR. (A) Schematic overview of the assay. A 281-bp-long DNA fragment containing the 5′ IR of Hsmar1 was used as the substrate for the reaction. The 5′ end of the IR is shown on a black background, the TA nucleotides bordering the element are in boldface, and the neighboring EcoRI site is underlined. Cleavage of one or both strands of the substrate DNA by the purified proteins results in phosphoryl-terminated DNA ends, which can later be ligated to 3′-hydroxyl-terminated single-stranded oligonucleotide linkers (gray lines) with T4 RNA ligase. The linkers tag the cleavage sites and enable amplification of the cleavage products by nested PCR. (B) Agarose gels with PCR products obtained with linker-specific primers (gray arrows in panel A) and primers specific for the upper or bottom strand of the substrate (black or white arrows in panel A, respectively). PCRs performed on EcoRI-digested DNA served as positive controls. (C) Distribution of cleavage positions identified after cloning and sequencing of the PCR products. The black triangles indicate cleavage sites introduced by MBP-Hsmar1-Ra, and the white triangles mark positions where MBP-SETMAR nicked the upper strand of the substrate DNA.
FIG. 7.
FIG. 7.
In vivo cleavage activity of SETMAR. (A) The agarose gel shows PCR products of excision assays on plasmid DNA from HeLa cells transfected with pHsmar1-neo and the helper plasmids indicated. The faint, smeary products around the expected size in lane 2 indicate loss of Hsmar1 transposons from the donor plasmids catalyzed by the SETMAR protein. (B) The overall structures of footprints from HeLa and CHO-K1 cells. The schematic view of the donor site is depicted above. Δ represents deletions, and the sequences in the white boxes are microhomologies at the junctions.
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